Protease is any enzyme that conducts proteolysis (or proteolytic degradation). Proteolysis is the breakdown of proteins into smaller polypeptides or amino acids, which generally occurs by the hydrolysis of a peptide bond. Protease activity is associated with the regulation of many cellular processes by activating or deactivating enzymes, transcription factors, and receptors. Proteolysis can therefore be a method of regulating biological processes by turning inactive proteins into active ones. For example, in the blood-clotting cascade an initial event triggers a cascade of sequential proteolytic activation of many specific proteases, resulting in blood coagulation. Further, the complement system of the immune response also involves a complex sequential proteolytic activation and interaction that result in an attack on invading pathogens.
Proteases generally fall into four main mechanistic classes: serine, cysteine, aspartyl and metalloproteases. In the active sites of serine and cysteine proteases, the eponymous residue is usually paired with a proton-withdrawing group to promote nucleophilic attack on a peptide bond. Aspartyl proteases and metalloproteases activate a water molecule to serve as the nucleophile, rather than using a functional group of the enzyme itself. However, the overall process of peptide bond cleaving is essentially the same for all protease classes and includes the protease recognizing a potential cleavage site (i.e., a protease recognition site having a sequence of amino acids recognizable to the protease as a potential cleavage site).
Proteolytic degradation by proteases has been detected by techniques using fluorescence, colorimetry, radioactivity, electrophoretic size separation, and electrochemistry. For example, Ionescu et al. (2006, Analytical Chemistry, 78:6327-31) describes the use of a glucose oxidase inner layer encased in a gelatinous polymer. The proteolytic degradation of the gelatinous polymer permitted the release of glucose oxidase, which in the presence of glucose generated detectable hydrogen peroxide. For example, the hydrogen peroxide could be electrooxidized to generate an amperometric signal at a sensor. However, this system suffers from having few gelatinous polymers useful for this assay. In particular, it would be necessary to identify a gelatinous polymer with a useful proteolytic cleavage recognition site.
Wu et al. (2012, Analyst, 137:4829-33) describes the use of a synthetic peptide containing a protease recognition sequence with a terminal biotin at one end, which was covalently bound to an electrode surface, to detect proteolytic degradation. The synthetic peptide was sequentially contacted with a sample containing a protease and Streptavidin-Alkaline Phosphatase. A reduction in signal of the phosphate cleaved substrate generated an electrochemical signal indicating the amount of protease activity.
Additionally, matrix metaloproteinases (MMPs) have been detected electrochemically by Shin et al. (2012, Analytical Chemistry, 85:220-7) by using a terminal Cys peptide covalently assembled onto a gold electrode surface, and having a methylene blue redox label at the other terminal end of the peptide. This synthetic oligopeptide is then subjected to proteolytic cleavage from a sample. The loss of signal predicts the level of protease activity present.
However, these existing assays for proteolytic enzymes lack the ability to generate a positive signal. A positive signal is needed for highly sensitive assays that can detect the lowest levels of disease markers with greater accuracy by amplifying the positive detection signal. Based on the foregoing, there remains a need for systems and methods to detect proteases as useful sensors for clinical diagnosis in a manner that may be amplified.